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Method for producing 5'-flavour development nucleic acid

A nucleotide and nucleotide sequence technology, applied in the field of producing 5'-taste nucleotides, can solve the problems of low conversion rate and long production cycle, and achieve the effects of low cost, cost saving and short cycle

Inactive Publication Date: 2007-10-31
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, this method still has the disadvantages of long production cycle and low conversion rate.

Method used

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  • Method for producing 5'-flavour development nucleic acid

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] 1. Construction of genetically engineered bacteria

[0034] Primers were designed according to the phoc gene sequence of Enterobacter aerogenes provided in GeneBank as follows:

[0035] Primer1: 5'-cg ggatcc catgaaaaagcgcgttctcgccctctg-3'(BamHI) (denoted as SEQ.ID.NO.5),

[0036] Primer2: 5'-ccg ctcgag cgatgacgttacttctgcgttttggcg-3' (XholI) (denoted as SEQ.ID.NO.6),

[0037] Perform PCR using the chromosomal DNA of Enterobacter aerogenes as a template: 95°C for 5 minutes, 30× (95°C for 30s, 55°C for 30s, 72°C for 60s), 72°C for 10 minutes. A PCR product with a length of about 760bp was obtained, which was ligated with the pBV220 vector with T4 DNA ligase after restriction enzyme digestion, and transformed into E.coliDH5α competent cells. The pBV220 vector general primer PCR and restriction enzyme digestion were used to identify the transformants to obtain the genetically engineered strain pBV220-phoc. The DNA sequencing result is SEQ.ID.NO.1:

[0038]atgaaaaagcgc...

Embodiment 2

[0055] 1. Mutation of phoc

[0056] Design the following primers:

[0057] Primer3: 5'-ctaacctcagcttcggcgacgtggc-3' (denoted as SEQ.ID.NO.7),

[0058] Primer4: 5'-gccacgtcgccgaagctgaggttag-3' (denoted as SEQ.ID.NO.8),

[0059] Primer5: 5'-gcatacctctaccggctgggccac-3' (denoted as SEQ.ID.NO.9),

[0060] Primer6: 5'-gtggcccagccggtagagaggtatgc-3' (denoted as SEQ.ID.NO.10),

[0061] Using primer1 / primer4, primer3 / primer6, primer5 / primer2 as primers, pBV220-phoc recombinant plasmid as template, PCR, the conditions are as follows: 95°C for 5min, 30×(95°C for 30s, 50°C for 30s, 72°C for 60s), 72°C for 10 minutes. The PCR products with a length of about 200bp, 200bp, and 300bp were respectively obtained, and after recovery, they were used as templates and primer1 / primer2 were used as primers for the second round of PCR. ℃ 60s), 72 ℃ 10min. A PCR product with a length of about 760bp was obtained, which was ligated with the pBV220 vector with T4 DNA ligase after restriction enzyme d...

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Abstract

The invention discloses a preparing method of 5' -nucleic acid in biochemical engineering technical, which comprises the following steps: 1, utilizing reconstructing DNA technique; constructing acid phosphatase expressing carrier; transforming bacillus coli; getting genetic engineering bacteria of high effective expressing acid phosphatase; 2, abrupt-changing acid phosphatase gene; sieving acid phosphatase with higher compatibility; 3, catalyzing nucleoside with acid phosphatase; synthesizing 5' -nucleic acid with phosphoric acid group donator. This product possesses short period and low cost, which is fit for commercial manufacture.

Description

technical field [0001] The invention belongs to the technical field of biochemical engineering, and in particular relates to a method for producing 5'-taste nucleotides by using highly active genetically engineered bacteria. Background technique [0002] 5'-nucleotides are often used as food additives and drug synthesis intermediates, of which 5'-inosine-5'-monophosphate (5'-IMP for short) and Guanosine-5'-monophosphate (Guanosine-5'-monophosphate , referred to as 5'-GMP) as a taste agent can be mixed with monosodium glutamate (MSG) to produce a synergistic effect, which can increase the freshness several times to dozens of times; in addition, they have synergistic effects on sweetness and meat flavor It has inhibitory effect on salty, sour, bitter, fishy and burnt taste, so it has been paid more and more attention and welcome in food industry production. [0003] The methods for industrial production of 5'-nucleotides mainly include ribonucleic acid (RNA) enzymatic hydroly...

Claims

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Application Information

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IPC IPC(8): C12N15/09C12N9/12C12P19/30
Inventor 梁胜华
Owner FUDAN UNIV
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